The major histocompatibility complex (MHC) of the mouse consists of a series of closely] linked loci on chromosome 17 which control cell membrane and serum glycoproteins involved in important functions of the immune system. Recombination over much of this area does not occur in a random manner, but in discrete areas termed "hotspots" of recombination. The major focus of this proposal is to study one such area of site-specific recombination, specifically that occurring in the second intron of the E-beta gene. The overall goal of this research is to add to our knowledge of the molecular mechanisms involved in eukaryote meiotic recombination. This grant application proposes to describe key nucleotide sequences located within the E-beta recombination area that are necessary for the cross-over event. Several areas of suspected involvement have been identified. These include retroposon sequence, a section of middle repetitive DNA with strong homology to the integration/excision sequence (Ins) of the mouse polyoma virus, and an area containing the tetrameric repeat AGGC. This project will be accomplished by the use of an In Vitro assay using plasmid/intron constructs. Two identical modules of each area suspected of being important in the recombination process will be cloned into pUC plasmids in such a manner allowing for the possibility of intramolecular recombination resulting in truncated plasmids. The constructs will also be designed in such a manner that an indicator gene (lacZ) will be excised out of the recombined plasmid. This will serve as a convenient marker to distinguish between non-recombined constructs and those in which a process of intramolecular recombination has taken place. Assays containing construct DNA will be carried out in the presence of testis cell extracts followed by transformation and plating. The relative frequency of lacZ- to lacZ+ colonies will then serve as an indicator of recombination efficiency. This research will lead to a clearer understanding as to the signals necessary for site-specific recombination.